Variants of human papillomavirus antigens

ABSTRACT

Variants of human papilloma virus (HPV) E6 and E7 able to elicit a humoral or cellular immune response against HPV in a host animal but not being cell-transforming in the host animal are disclosed, and are useful in treatment or prevention of diseases or conditions involving HPV.

FIELD OF THE INVENTION

This invention relates generally to variants of human papilloma virus(HPV) antigens, and in particular it relates to non-transformingvariants of HPV antigens which are suitable for use in vaccines. Theinvention also extends to vaccine compositions which include thesevariants of HPV antigens as active immunogens, as well as to methods ofuse of these variants to elicit an immune response against HPV.

BACKGROUND OF THE INVENTION

Papillomaviruses are small DNA viruses that infect a variety of animalspecies. Some are associated with the development of malignancies intheir natural hosts. Over 60 types of human papillomavirus (HPV) havebeen identified. These infect humans at a variety of body locations andare responsible for common skin warts, laryngeal papillomas, genitalwarts and other wart-like lesions. Genital HPV infections areparticularly common and a number of HPV types, but most frequently types6, 11, 16 and 18, infect the genital tract in both men and women. Inwomen, HPVs infect various portions of the genital tract including thecervix.

Genital HPVs are a significant clinical problem. HPV infection of theano-genital region is now regarded as the most common form of viralsexually-transmitted disease (STD). The viruses cause genital infectionswhich become manifest in one of three ways:

i clinical infection, where gross genital warts are visible;

ii subclinical infection, where viral lesions are not obvious but aredetectable using special viewing techniques; and

iii latency, where the only sign of infection is the presence of HPVDNA.

Subclinical infections are common. It is estimated that 2 to 4% ofPapanicolaou (Pap.) smears show evidence of HPV. Latent infections areeven more frequent and the majority of adults harbour one or more typesof genital HPV.

Carcinoma of the uterine cervix (CaCx) is a common cancer in women. Twoforms of cervical cancer are recognised; squamous cell carcinoma (SCC)is by far the most frequent representing about 90% of observed cases;adenocarcinoma, a cancer of the secretion cells, accounts for about 10%.Cancer of the cervix develops through pre-cancerous intermediate stagesto invasive forms (the carcinoma) which can become life threatening. Thepre-cancerous stages of increasing severity are known as cervicalintraepithelial neoplasia (CIN) grades 1 to 3. Over a 20 year periodabout 40% of the untreated CIN3 patients develop invasive cancer, theincreasingly serious forms of which are known as stage I to IV. Invasivecancer frequently leads to death.

Cervical cancer in both its pre-cancerous and invasive stages is one ofthe few cancers for which a highly reliable and relatively cheapscreening method is available. The Papanicolaou (Pap.) smear involvescytological examination of cervical scrapes to test for the presence ofabnormal cervical cells which are indicative of pre- or invasive cancer.Detection of abnormalities leads to further investigation and treatmentif necessary.

To be effective at reducing the number of cervical cancers and resultantdeaths, Pap. smear screening is undertaken on a mass scale and ideallyincludes all women of sexually-active age. Detection and subsequenttreatment of CIN has a very high success rate in the prevention ofinvasive cancer, while early detection of the latter can have a markedeffect on mortality.

Most developed countries have highly developed Pap. smear screeningprograms which have resulted in a 30% drop in age-specific mortality dueto CaCx between 1960 and 1980. However, apart from the Scandinaviancountries, few developed countries screen more than 50 to 60% of women,allowing CaCx and resultant deaths to remain a significant problem. Inthe developing world the situation is even worse as few organisedscreening programs exist, resulting in 400,000 new cases of invasivecancer annually in these countries.

As outlined earlier, a variety of types of HPV cause genital infectionsin humans, although four types (6, 11, 16, 18) predominate. Evidencecollected over the past 15 years strongly suggests that several of theHPVs are associated with the development of cervical cancer. Indeed manyresearchers have concluded that specific HPV types are the essentialaetiologic factor responsible for the development of many of thecancers.

Infection with HPV-16 and HPV-18 has been associated with thedevelopment of cancer of the cervix. It has been postulated that HPVacts as an initiator in cervical carcinogenesis and that malignanttransformation depends on interaction with other factors. Infectionswith HPV-6 and HPV-11 have been associated with the development ofgenital warts. The incidence of HPV infection appears to be increasingas shown by a large increase recently in patient visits related togenital HPV infections in both males and females and the presence of HPVin Pap. smears of some women under 30 years of age.

The nature of HPV-16 in particular and papilloma viruses in general hasbeen well studied recently. HPV-16 contains a 7904 bp double-strandedDNA genome (Siedorf, K. et al., Virology (1985) 145:181-185). The capsidis 50 nm and contains 72 capsomers (Klug, A., J. Mol. Biol. (1965)11:403-423). U.S. Pat. No. 4,777,239 discloses a series of 17 syntheticpeptides which are said to be capable of raising antibodies to HPV-16and thus may be useful for diagnostic purposes. In addition, EuropeanPatent 0 412 762 discloses polypeptides which are antagonists of thebiochemical interaction of the HPV E7 protein and the retinoblastomagene (RBG) protein, and which are said to be useful in the treatment ofgenital warts and cervical cancer.

The DNAs of several papilloma viruses have been sequenced, includingseveral HPV types, bovine papillomavirus (BPV) and cottontail rabbitpapillomavirus (CRPV). All of these display similar patterns ofnucleotide sequence with respect to open reading frames. The openreading frames can be functionally divided into early regions (E) andlate regions (L); the E regions are postulated to encode proteins neededfor replication and transformation; and the L regions to encode theviral capsid proteins (Danos, O., et al., J. Invest. Derm. (1984)83:7s-11s).

Two HPV encoded proteins, E6 and E7, are thought to be involved in thepathogenesis of HPV-induced abnormal cell proliferation (reviewed inStoppler et al., Intervirology, (1994) 37:168-179). The amino acidsequences of the HPV-16 E6 and E7 proteins as deduced from the nucleicacid sequence are shown in Siedorf et al., Virology, (1985) 145:181-185.

The HPV genes encoding the E6 and E7 proteins are invariably expressedin tissue or tumor cells obtained from cervical cancers associated withHPV infection. In addition, the HPV E6 and E7 genes derived from theHPV-16 strain are capable of inducing epithelial cell transformation incell culture without the presence of other HPV genes. These observationsindicate that at least part of the stimulation of cell proliferationcaused by HPV infection is due to the E6 and E7 viral proteins.

The HPV E6 and E7 proteins are believed to be effective immunologicaltargets for tumour regression. As described above, however, the E6 andE7 genes are known to "transform" cells possibly by the action of theirprotein products in interfering with cellular proteins involved in thecontrol cell growth. Accordingly, if even minute traces of DNA encodingthe E6 and E7 proteins were to be present in a vaccine preparation, thiscould cause that vaccine preparation to initiate irreversibletransformation events in the cells of a recipient of the vaccinepreparation. It is an object of the present invention to providenon-transforming variants of the HPV E6 and E7 proteins which are ableto induce in a host animal (particularly a human) a range of humoral andcellular immune responses, and which are therefore suitable for use inthe production of vaccines for the prevention, prophylaxis, therapy andtreatment of HPV-induced diseases or other conditions which wouldbenefit from inhibition of HPV infection.

In the work leading to the present invention, it has been recognisedthat there are four ways to induce immune responses to E6 and/or E7proteins:

(i) use whole proteins (this introduces the possibility thatcontaminating DNA may be associated with the proteins);

(ii) use point mutants (this can lead to reversion to native protein,which requires multiple mutations to avoid; in addition, any pointmutation leads to loss of potentially vital epitopes);

(iii) use specific peptides (this requires a very large number ofpeptides, the identification of which is very complex, to make a vaccineof broad utility); and

(iv) use variants such as fusions and combinations of deletion mutants(this method has none of the above limitations).

In addition to the cell transforming properties of the E7 proteinitself, fusions of this protein with β-galactosidase have also beenshown to be cell-transforming (Fujikawa et al., Virology, 204, 789-793,1994). Accordingly, it could not be predicted that fusions of E6 and/orE7 moieties, either full length or non-full length, would not also becell-transforming.

SUMMARY OF THE INVENTION

In one aspect the present invention provides as an isolated protein, avariant of the HPV E6 or E7 protein which variant is able to elicit ahumoral and/or cellular immune response against HPV in a host animal butwhich is not cell-transforming in the host animal.

In another aspect, the present invention provides a method for elicitingan immune response against HPV in a host animal, which method comprisesadministering to the host animal an effective amount of a variant of theHPV E6 or E7 protein which variant is able to elicit a humoral and/orcellular immune response against HPV in a host animal but which is notcell-transforming in the host animal.

In yet another aspect, the present invention provides a vaccinecomposition for use in eliciting an immune response against HPV in ahost animal, which comprises a variant of the HPV E6 or E7 protein whichvariant is able to elicit a humoral and/or immune response against HPVin a host animal but which is not cell-transforming in the host animal,and optionally an adjuvant, together with a pharmaceutically acceptablecarrier and/or diluent.

The invention also extends to the use of a variant of the HPV E6 or E7protein which variant is able to elicit a humoral and/or cellular immuneresponse against HPV in a host animal but which is not cell-transformingin the host animal, and optionally an adjuvant, in eliciting an immuneresponse against HPV in a host animal.

Throughout this specification, unless the context requires otherwise,the word "comprise", or variations such as "comprises" or "comprising",will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a)-(d) show domain maps for the different proteins referencedherein.

FIG. 2 shows amounts of fusion protein produced in transformed cellsfollowing IPTG induction in Example 1.

FIG. 3 shows a western blot of a protein described in Example 2.

FIG. 4(a) shows a coomassie stained gel indicating amounts of fusionprotein produced in transformed cells following IPTG induction inExample 3(i).

FIG. 4(b) is a western blot confirming the identity of the protein ofExample 3(i).

FIG. 5(a) shows a coomassie stained gel indicating amounts of fusionprotein produced in transformed cells following IPTG induction inExample 3(ii).

FIG. 5(b) is a western blot confirming the identity of the protein ofExample 3(ii).

FIGS. 6(a) and (b) show coomassie stained gel and western blot,respectively, confirming purity and identity of the product of Example5.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, references to the variant of this invention as being"not cell-transforming in the host animal" mean that thecell-transforming property of the "parent" or wild-type HPV E6 or E7protein has been reduced, and preferably effectively eliminated, in thevariant. In particular, these references indicate that thiscell-transforming property has been significantly reduced in comparisonwith wild-type E6 or E7 protein in appropriate test systems.

It will be appreciated that where the non-transforming variant HPV E6 orE7 protein of this invention is produced by expression of an appropriateencoding recombinant DNA molecule, the nature of that encoding DNA,unlike the wild-type E6 or E7 genes, would not have the potential toinitiate irreversible transformation events in the cells of the hostanimal.

The variant HPV E6 or E7 proteins of the present invention include, butare not limited to, deletion mutants of the wild-type E6 or E7 proteinsin the form of non-full length fragments of the wild-type proteins, aswell as fusion proteins in which E6 and/or E7 moieties are fused,optionally with a linkage of from 1 to 50, preferably a short linkage offrom 1 to 20, and more preferably from 1 to 5, amino acid residuesbetween the E6 and/or E7 moieties. The E6 and/or E7 moieties in such afusion protein may comprise the full wild-type E6 or E7 proteins, oralternatively they may comprise non-full length fragments of thewild-type proteins. The fusion proteins may also comprise other moietiesfused or otherwise coupled thereto, for example moieties to assist inpurification of the fusion protein (for example, aglutathione-S-transferase or GST moiety or hexa-His moiety) or toenhance the immunogenicity of the fusion protein (for example anadjuvant such as diphtheria or cholera toxin or a non-toxic derivativethereof such as the holotoxoid or B sub-unit of cholera toxin).

The term "non-full length fragment" is used herein to describepolypeptides which may for example comprise deletion mutants of the E6or E7 proteins corresponding to at least 50%, more preferably 60-70%,and even 80-90% of the full-length E6 or E7 protein sequence. By way ofexample only, the fragments may be deletion mutants corresponding to theN-terminal or C-terminal two-thirds of the E6 or E7 proteins.

Suitable non-full length fragments, and fusion proteins which comprisethe E6 and/or E7 proteins or non-full length fragments thereof, asdescribed above may be readily produced by techniques which are wellknown in the art and which are described by way of example below. Itwill be appreciated by persons skilled in this art that variant HPV E6or E7 proteins as described above including fusion proteins whichcomprise various combinations of the E6 and/or E7 moieties may bereadily produced using these. known techniques, and then tested usingroutine methods to establish whether the resultant fusion protein orother variant protein meets the criteria of the present invention, thatis whether it is able to elicit a humoral and/or cellular immuneresponse in a host animal but is not cell-transforming in the hostanimal.

Preferably, the host animal is a human, however the host animal may alsobe a non-human mammal.

The present invention is particularly, but not exclusively, directed tovariants of the E6 or E7 proteins of the HPV-16 and HPV-18 genotypes,however it will be appreciated that the invention extends to variants ofthe corresponding proteins in other HPV genotypes, particularly theHPV-6 and HPV-11 genotypes which are causative agents of condylomataacuminta, and other genotypes which have oncogenic potential of a typesimilar to HPV-16 and HPV-18.

Previous work in this area has shown that vaccination of rats with liveviral vectors expressing HPV E6 or E7 proteins leads to rejection oftransplanted E7-bearing tumour cells (Meneguzzi et al., Virology,181:62-69, 1991), while vaccination of cattle with an adjuvanted HPV E7vaccine leads to accelerated rejection of tumours induced by bovinepapillomavirus (Campo, Cancer Cells, 3:421-426, 1991).

The variant HPV E6 or E7 proteins of the present invention are providedas isolated proteins, that is they are substantially free of other HPVproteins, and find particular utility for the treatment of genitalwarts, cervical cancer or other conditions caused by HPV in man. Thevariant proteins can be included in pharmaceutical compositions for thetreatment or prevention of diseases involving HPV as well as the otherconditions discussed above.

The variant HPV E6 or E7 proteins of the invention may be used to raiseantibodies andlor induce cellular immune responses, either in subjectsfor which protection against infection by HPV is desired, i.e. asprophylactic vaccines, or to heighten the immune response to an HPVinfection already present, i.e. as therapeutic vaccines. They also canbe injected into production species to obtain antisera. In lieu of thepolyclonal antisera obtained in the production species, monoclonalantibodies may be produced using the standard methods or by more recentmodifications thereof by immortalising spleen or otherantibody-producing cells for injection into animals to obtainantibody-producing clones. The polyclonal or monoclonal antibodiesobtained, corrected if necessary for species variations, can also beused as therapeutic agents.

Direct administration of the variant proteins to a host can confereither protective immunity against HPV or, if the subject is alreadyinfected, a boost to the subject's own immune response to moreeffectively combat the progress of the HPV induced disease.

The magnitude of the prophylactic or therapeutic dose of a variant HPVE6 or E7 protein of this invention will, of course, vary with the groupof patients (age, sex, etc.), the nature or the severity of thecondition to be treated and with the particular variant protein and itsroute of administration. In general, the weekly dose range for use lieswithin the range of from about 0.1 to about 5 μg per kg body weight of amammal.

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dosage of a variantprotein of this invention. For example, oral, rectal, vaginal, topical,parenteral, ocular, nasal, sublingual, buccal, intravenous and the likemay be employed. Dosage forms include tablets, troches, dispersions,suspensions, solutions, capsules, creams, ointments, suppositories,aerosols and the like. Said dosage forms also include injected orimplanted slow releasing devices specifically designed for this purposeor other forms of implants modified to additionally act in this fashion.

If the variant proteins are to be administered as vaccines, they areformulated according to conventional methods for such administration tothe subject to be protected. If the antibodies are to be used fortherapeutic purposes, it is generally desirable to confer speciescharacteristics upon them compatible with the subject to be treated.Accordingly, it is often desirable to prepare these antibodies inmonoclonal form since fusion with suitable partners is capable ofconferring the desired characteristics on the secreted monoclonals.

The variant proteins may be delivered in accordance with this inventionin ISCOMS™ (immune stimulating complexes), liposomes or encapsulated incompounds such as acrylates or poly(DL-lactide-co-glycoside) to formmicrospheres. The variant proteins may also be incorporated into oilyemulsions and delivered orally.

Other adjuvants, as well as conventional pharmaceutically acceptablecarriers, excipients, buffers or diluents, may also be included in thevaccine compositions of this invention. Generally, a vaccine compositionin accordance with the present invention will comprise animmunologically effective amount of the variant HPV E6 or E7 protein,and optionally an adjuvant, in conjunction with one or more conventionalpharmaceutically acceptable carriers and/or diluents. An extensivethough not exhaustive list of adjuvants can be found in Coulter and Cox,"Advances in Adjuvant Technology and Application", in Animal ParasiteControl Utilizing Biotechnology, Chapter 4, Ed. Young, W. K., CRC Press,1992. As used herein "pharmaceutically acceptable carriers and/ordiluents" include any and all solvents, dispersion media, aqueoussolutions, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents and the like. The use of such media andagents for pharmaceutical active substances is well known in the art andis described by way of example in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, U.S.A.

In practical use, a variant protein of this invention can be combined asthe active ingredient in intimate admixture with a pharmaceuticalcarrier according to conventional pharmaceutical compounding techniques.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g. oral or parenteral(including intravenous and intra-arterial). In preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water glycols, oils, alcohols,flavouring agents, preservatives, colouring agents and the like in thecase of oral liquid preparations, such as, for example, suspensions,elixirs and solutions; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like in the case of oral solidpreparations such as, for example, powders, capsules and tablets.Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe sugar-coated or enteric-coated by standard techniques.

In addition to the common dosage forms set out above, the variantproteins of this invention may also be administered by controlledrelease means and/or delivery devices, including by way of example, thecontrolled release preparations disclosed in International PatentSpecification No. PCT/AU93/00677 (Publication No. WO 94/15636).

Pharmaceutical compositions of the present invention suitable for oralor parenteral administration may be presented as discrete units such ascapsules, cachets or tablets each containing a predetermined amount ofthe active ingredient, as a powder or granules or as a solution or asuspension in an aqueous liquid, a non-aqueous liquid, an oil-in-wateremulsion or a water-in-oil liquid emulsion. Such compositions may beprepared by any of the methods of pharmacy but all methods include thestep of bringing into association the active ingredient with the carrierwhich constitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or fmely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation.

Further features of the present invention are more fully described inthe following Example(s). It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention, and should not be understood in any way as arestriction on the broad description of the invention as set out above.

EXAMPLE 1 Cloning and Expression of GST E6/E7 Fusion Protein

A molecule consisting of HPV-16 E6 and E7 sequences as an "in-frame"fusion was created as follows. A clone of HPV-16 DNA containing both E6and E7 genomic sequences served as the template for separate PCRamplification of E6 and E7 using oligonucleotides:

(a) (5')CGCTCGAGAGATCTCATATGCACCAAAAGAGAACTGC(3') and

(b) (5')CGCCCGGGCAGCTGGGTTTCTCTACGTG(3') for E6; and

(c) (5')CGCCCGGGATGCATGGAGATACACCTACATTGCATG(3') and

(d) (5')CGGTCGACGGATCCTGGTTTCTGAGAACAGATGGG(3') for E7.

A SmaI recognition site at the 3' end of Et and the 5' end of E7facilitated the fusion and introduced two additional amino acids(proline and glycine) between E6 and E7. Additional restriction enzymerecognition sites at the 5' and 3' boundaries of the fusion molecule(introduced in the oligonucleotides) aided in subsequent cloningprocedures.

The fused E6/E7 sequence was cloned as a BglII-BamH1 fragment intovector pDS56 (Stuber et al., EMBO J., (1984) 3:3143-3148) which providedan in-frame 3' hexa-his(hh) sequence. From this, E6/E7hh was removed asa EcoRI/Hind III fragment and subdloned into pGEM 7+3, which was createdby inserting the BamH1/HindIII portion of the pGEM3-Zf(+)(Promega)polylinker into the BamH1/HindIII site of the multiple cloning site ofthe pGEM7-Zf(+)(Promega) vector. E6/E7hh was then removed from pGEM7+3as a EcoRI/Sal I fragment and inserted into the multiple cloning site ofpGEX-4T-1 (Pharmacia) to produce pGEX-4T-1 E6/E7hh. This plasmid wasused to transform a variety of E. coli strains including TOPP2(Stratagene) and BL21 (Amrad/Pharmacia). Both types of transformed cellsproduced a significant amount of fuision protein following IPTGinduction (FIG. 2). The fusion protein (GST E6/E7hh representedschematically in FIG. 1a) was in the expected size range of around 60kDa. The identity of the protein was confirmed by Western blots probedwith two monoclonal antibodies directed against E7 (LHIL.16E7.8F andLHIL.16E7.6D, Tindle et al. Journal of General Virology, (1990)71:1347-1354) (FIG. 3).

EXAMPLE 2 Cloning and Expression of E6/E7 Fusion Protein

In order to express E6/E7 hh as protein lacking GST, a termination codonwas introduced into pGEX-4T-1 E6/E7hh at a unique BalI site 3' to, andin-frame with, the GST translation initiation codon using thephosphorylated linker TGCTCTAGAGCA. After transforming E. coli strainBL21 with this new plasmid ([GST] E6/E7hh) a significant amount ofprotein (E6/E7hh, represented schematically in FIG. 1b) was producedfollowing IPTG induction at a size of approximately 33 kD whichcorresponds to the size expected of a E6/E7hh fusion protein (FIG. 2).Identity of this protein was confirmed by Western blot using the samemonoclonal antibodies as in Example 1 (FIG. 3).

EXAMPLE 3 Cloning and Expression of Deleted (Non-full Length) Forms ofE6 and E7

(i) Construction of ΔE6C/ΔE7N

Full length E6/E7 in pGEM3(Promega) served as a template for PCRamplification of deleted forms of E6/E7 using oligonucleotides5'GCGCGAATTCTATTAAGGAGCCCGGGATGGGGAATCCATATGCTGTAT3' and5'CGCGAGATCTCCGAAGCGTAGAGTCACACTTG3'.

The resulting truncation of E6/E7 lacking sequences (189 bp) at the Nterminal of E6 and C terminal of E7 (96 bp) was subcloned into pGEX-4T-1containing a termination codon in the GST sequence to produce [GST]ΔE6C/ΔE7Nhh. This plasmid was used to transform E. coil strain BL21.Transformed cells expressed a significant amount of fusion protein(ΔE6C/ΔE7Nhh, represented schematically in FIG. 1c) following IPTGinduction (FIG. 4a) producing a protein of the approximate expected size(20 kD). The identity of this protein was confirmed by Western blotusing the same monoclonal antibodies as in Example 1 (FIG. 4b).

(ii) Construction of ΔE7C/ΔE6N

Using oligonucleotides (a) in Example 1 and5'CGCCCGGGTAATGTTGTTCCATACAAACTA3' an N-terminal representation of E6comprising 285 bp was amplified from the same HPV-16 clone utilised inExample 1. As well, oligonucleotides 5'CGCCCGGGGAGGAGGAGGATGAAATAGATG3'and (d) in Example 1 were used to produce a 198 bp C-terminal E7sequence. These were each blunt cloned into pGEM7-Zf(+) (Promega). Afusion cassette was formed by restricting the E6 clone with KpnI/BglIIand inserting the E7 sequence upstream as a KpnI/BamHI fragment. Thisfused sequence was then reamplified with SmaI and BglII cloning sitesfor insertion into pGEX-4T-1 containing a termination codon in the GSTsequence to produce [GST] ΔE7C/ΔE6Nhh. After transformation into E. coliBL21, protein production was assayed by PAGE followed by Coomassiestaining and Western blotting (FIGS. 5a and 5b). A protein (ΔE7C/ΔE6Nhh,represented schematically in FIG. 1d) of the expected size (20 kD) wasevident on Western blots.

EXAMPLE 4 DNA Sequencing of E6/E7 Full Length and Deletion Constructs

E6/E7 constructs were sequenced in both directions by the dideoxy methodusing primers that generated overlapping sequence information. The ^(T7)Sequencing™ Kit (Pharmacia) was used to generate ³⁵ S-labelledchain-terminated fragments which were analysed on a Sequi-Gen™ (Biorad)electrophoretic gel apparatus. The DNA and corresponding amino acidsequences for E6/E7hh (PPV162.DNA), ΔE6C/ΔE7Nhh (CAD600.SEQ) andΔE7C/ΔE6Nhh (C620.TXT) are set out below.

    5' ATG CAC CAA AAG AGA ACT GCA ATG TTT CAG GAC CCA CAG GAG CGA CCC AGA        AAG                                                                           --                                                                               Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg        Lys                                                                              TTA CCA CAG TTA TGC ACA GAG CTG CAA ACA ACT ATA CAT GAT ATA ATA TTA        GAA                                                                           --                                                                               Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu        Glu                                                                              TGT GTG TAC TGC AAG CAA CAG TTA CTG CGA CGT GAG GTA TAT GAC TTT GCT        TTT                                                                           --                                                                               Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala        Phe                                                                              CGG GAT TTA TGC ATA GTA TAT AGA GAT GGG AAT CCA TAT GCT GTA TGT GAT        AAA                                                                           --                                                                               Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp        Lys                                                                              TGT TTA AAG TTT TAT TCT AAA ATT AGT GAG TAT AGA CAT TAT TGT TAT AGT        TTG                                                                           --                                                                               Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser        Leu                                                                              TAT GGA ACA ACA TTA GAA CAG CAA TAC AAC AAA CCG TTG TGT GAT TTG TTA        ATT                                                                           --                                                                               Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu        Ile                                                                              AGG TGT ATT AAC TGT CAA AAG CCA CTG TGT CCT GAA GAA AAG CAA AGA CAT        CTG                                                                           --                                                                               Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His        Leu                                                                              CAG AAA AAG CAA AGA TTC CAT AAT ATA AGG GGT CGG TGG ACC GGT CGA TGT        ATG                                                                           --                                                                               Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys        Met                                                                              TCT TGT TGC AGA TCA TCA AGA ACA CGT AGA GAA ACC CAG CTG CCC GGG ATG        CAT                                                                           --                                                                               Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Pro Gly Met        His                                                                              GGA GAT ACA CCT ACA TTG CAT GAA TAT ATG TTA GAT TTG CAA CCA GAG ACA        ACT                                                                           --                                                                               Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr        Thr                                                                              GAT CTC TAC TGT TAT GAG CAA TTA AAT GAC AGC TCA GAG GAG GAG GAT GAA        ATA                                                                           --                                                                               Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu        Ile                                                                              GAT GGT CCA GCT GGA CAA GCA GAA CCG GAC AGA GCC CAT TAC AAT ATT GTA        ACC                                                                           --                                                                               Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val        Thr                                                                              TTT TGT TGC AAG TGT GAC TCT ACG CTT CGG TTG TGC GTA CAA AGC ACA CAC        GTA                                                                           --                                                                               Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His        Val                                                                              GAC ATT CGT ACT TTG GAA GAC CTG TTA ATG GGC ACA CTA GGA ATT GTG TGC        CCC                                                                           --                                                                               Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys        Pro                                                                              ATC TGT TCT CAG AAA CCA AGA TCT CAT CAC CAT CAC CAT CAC TAA 3'                Ile Cys Ser Gln Lys Pro Arg Ser His His His His His His ***                File: CAD600.SEQ                                                              Range: 1- 519 Mode: Normal                                                    Codon: Universal                                                              54                                                                            5' ATG GGG AAT CCA TAT GCT GTA TGT GAT AAA TGT TTA AAG TTT TAT TCT AAA        ATT                                                                           --                                                                               Met Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys        Ile                                                                              AGT GAG TAT AGA CAT TAT TGT TAT AGT TTG TAT GGA ACA ACA TTA GAA CAG        CAA                                                                           --                                                                               Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln        Gln                                                                              TAC AAC AAA CCG TTG TGT GAT TTG TTA ATT AGG TGT ATT AAC TGT CAA AAG        CCA                                                                           --                                                                               Ile Asn Cys Gln Lys Pro Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg        Cys                                                                              CTG TGT CCT GAA GAA AAG CAA AGA CAT CTG GAC AAA AAG CAA AGA TTC CAT        AAT                                                                           --                                                                               Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His        Asn                                                                              ATA AGG GGT CGG TGG ACC GGT CGA TGT ATG TCT TGT TGC AGA TCA TCA AGA        ACA                                                                           --                                                                               Ile Arg Gly Arg Trp Thr Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg        Thr                                                                              CGT AGA GAA ACC CAG CTG CCC GGG ATG CAT GGA GAT ACA CCT ACA TTG CAT        GAA                                                                           --                                                                               Arg Arg Glu Thr Gln Leu Pro Gly Met His Gly Asp Thr Pro Thr Leu His        Glu                                                                              TAT ATG TTA GAT TTG CAA CCA GAG ACA ACT GAT CTC TAC TGT TAT GAG CAA        TTA                                                                           --                                                                               Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln        Leu                                                                              AAT GAC AGC TCA GAG GAG GAG GAT GAA ATA GAT GGT CCA GCT GGA CAA GCA        GAA                                                                           --                                                                               Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala        Glu                                                                              CCG GAC AGA GCC CAT TAC AAT ATT GTA ACC TTT TGT TGC AAG TGT GAC TCT        ACG                                                                           --                                                                               Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser        Thr                                                                              CTT CGG AGA TCT CAT CAC CAT CAC CAT CAC TAA 3'                             --                                                                               Leu Arg Arg Ser His His His His His His ***                                File: C620.TXT                                                                Range: 1- 519 Mode: Normal                                                    Codon Table: Universal                                                        5' ATG GAG GAG GAT GAA ATA GAT GGT CCA GCT GGA CAA GCA GAA CCG GAC AGA        GGC                                                                           --                                                                               Met Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg        Ala                                                                              CAT TAC AAT ATT GTA ACC TTT TGT TGC AAG TGT GAC TCT ACG CTT CGG TTG        TGC                                                                           --                                                                               His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu        Cys                                                                              GTA CAA AGC ACA CAC GTA GAC ATT CGT ACT TTG GAA GAC CTG TTA ATG GGC        ACA                                                                           --                                                                               Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly        Thr                                                                              CTA GGA ATT GTG TGC CCC ATC TGT TCT CAG AAA CCA GGA TCT CAT ATG CAC        CAA                                                                           --                                                                               Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Gly Ser His Met His        Gln                                                                              AAG AGA ACT GCA ATG TTT CAG GAC CCA CAG GAG CGA CCC AGA AAG TTA CCA        CAG                                                                           --                                                                               Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro        Gln                                                                              TTA TGC ACA GAG CTG CAA ACA ACT ATA CAT GAT ATA ATA TTA GAA TGT GTG        TAC                                                                           --                                                                               Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val        Tyr                                                                              TGC AAG CAA CAG TTA CTG CGA CGT GAG GTA TAT GAC TTT GCT TTT CGG GAT        TTA                                                                           --                                                                               Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp        Leu                                                                              TGC ATA GTA TAT AGA GAT GGG AAT CCA TAT GCT GTA TGT GAT AAA TGT TTA        AAG                                                                           --                                                                               Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu        Lys                                                                              TTT TAT TCT AAA ATT AGT GAG TAT AGA CAT TAT TGT TAT AGT TTG ATA GGA        ACA                                                                           --                                                                               Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly        Thr                                                                              ACA TTA AGA TCT CAT CAC CAT CAC CAT CAC TAA 3'                             --                                                                               Thr Leu Arg Ser His His His His His His ***                            

EXAMPLE 5 Immunogenicity of E6/E7hh Protein

A. Purification of E6/E7hh

E. coli cells (strain BL21) containing the [GST] E6/E7hh plasmid wereinduced using 0.1-0.5 mM IPTG and harvested 3-4 hours after induction.The cells were pelleted by low speed centrifugation and inclusion bodiescontaining the E6/E7hh protein isolated by sonication andcentrifugation. The inclusion pellet was solubilised in 7M Urea or 6MGuanidine HCl and subjected to nickel chelate column chromatography(Porath et.al., Biochemistry22, 1621-1630, 1983). Protein was elutedusing either an increasing gradient of imidazole or a decreasing pHgradient, and fractions containing E6/E7hh pooled and dialysed against25 mM Tris, 0.5M NaCl, 1% NOG, 10 mM DTT pH7.5. The identity and purityof the dialysed product was determined by Coomassie stainedpolyacrylamide gel electrophoresis and Western blot using the monoclonalantibodies referred to in Example 1 (FIGS. 6a and 6b).

B. Immunogenicity of E6/E7hh

On day 0, two groups of 5 C57BL/6 mice (8 weeks old, female) wereinoculated subcutaneously at the base of the tail with 0.1 mL of aformulation containing 6 μg ISCOMATRIX™, 19 μg E6/E7hh (purified as inA. above) in PBS pH7.2. A second dose of the formulation wasadministered at day 14 to group 1, and at day 17 to group 2. At day 21and 24, mice in groups 1 and 2 (respectively) were bled. Serum antibodyresponses to E6/E7hh were then measured using the following solid phaseEIA:

Nunc MaxiSorp EIA plates were coated with E6/E7hh by incubating 0.1mL/well for 2 hours at 37° of a 10 μg/mL solution in 4M urea in 50 mMcarbonate buffer, pH 9.5. The liquid was removed, and the plates werefurther incubated at 37° for 1 hour with 0.2 mL/well of 1 mg/mL caseinin PBS pH 7.2. After 6 washes, 0.1 mL/well of test serum (diluted in PBSpH 7.2, 1 mg/mL casein, 0.5% Tween 20, 0.002% alphazurine A) was added,and the plates incubated for 1 hour at 37°. The plates were then againwashed 6× with PBS pH 7.2, 0.5% Tween 20. To detect bound antibody, 0.1mL of 0.1 μg/ml KPL horseradish peroxidase-labelled goat anti-mouseIgG+IgM (H and L chain specific) in PBS pH 7.2, 1 mg/mL casein, 0.5%Tween 20, 0.002% alphazurine A was added to each well. The plates wereincubated for 1 hour at 20°, washed 6× with PBS pH 7.2, 0.5% v/v Tween20, then 0.1 mL of enzyme substrate (3,3',5,5'tetramethylbenzidine/H₂ O₂formulation, purchased from KPL) was then added. After 10 minutesincubation at 20°, the reaction was stopped by addition of 50 μl of 0.5MH₂ SO₄. The coloured product was then measured at 450 nm in a verticalbeam spectrophotometer. Titres were expressed as the reciprocal of theserum dilution resulting in an optical density value of 0.1.

Table 1 shows that all mice of both groups 1 and 2 produced asignificant response following immunization. Titres ranged from 3.17 to5.66 (expressed in the log₁₀ of the reciprocal dilution resulting in theoptical density of 0.1 in the solid phase EIA described above).Pre-existing antibody levels were low or undetectable (measured in seraobtained on day 0 immediately prior to inoculation).

Clearly, the E6/E7hh fusion protein is highly immunogenic whenadministered to mice by this procedure.

As well, E6/E7hh was found to produce specific delayed-typehypersensitivity (DTH) following one dose of a formulation containingE6/E7hh plus ISCOM™ adjuvant. The mice produced specific DTH responsesto both E6 and E7 when challenged in the ear with small doses ofpurified GST-E6 or GST-E7 proteins.

                                      TABLE 1                                     __________________________________________________________________________    Log dilution to 0.1 OD.                                                       __________________________________________________________________________    Group/mouse                                                                          1/1                                                                              1/2                                                                              1/3                                                                              1/4                                                                              1/5                                                                              2/1                                                                              2/2                                                                              2/3                                                                              2/4                                                                              2/5                                         pre-bleed                                                                            <2 <2 <2 <2 <2 <2 2.06                                                                             <2 <2 <2                                          final bleed                                                                          3.66                                                                             5.66                                                                             3.23                                                                             3.19                                                                             3.79                                                                             4.19                                                                             4.21                                                                             3.71                                                                             5.55                                                                             3.17                                        __________________________________________________________________________

EXAMPLE 6 Transformation Studies of E6/E7 Gene Construct

An E6/E7 fusion DNA construct was subcloned into the multiple cloningsite of plasmid vector pJ4Ω (Wilkinson et al., J. Exp. Med. (1988)167:1442-58) as a BamHI fragment to produce pJ4Ω E6/E7. For comparisonpurposes pJ4Ω vectors containing HPV16 E6 (pJ4Ω E6) and HPV16 E7(pJ4ΩE7) ORFs were used. Where neomycin selection was required, thepcDNA3 vector (Invitrogen) containing a neomycin resistance marker wasutilised. These plasmids were amplified in E. coli and plasmid DNAextracted by alkaline lysis and purified on resin (Qiagen) eluted,ethanol precipitated and resuspended in H₂ O. DNA quantity and puritywas determined by spectrophotometric measurement at 260 and 280 nm. DNAintegrity was checked by electrophoresis in 1% agarose gels and ethidiumbromide staining. Target cells for transformation were mouse NIH 3T3cells (CSL Biosciences). The cells were routinely propagated on MinimalEssential Medium (Eagle) supplemented with non-essential amino acids, 2mM glutamine and 10% foetal bovine serum (growth medium).

Transfection of NIH 3T3 cells with plasmid DNA was carried outessentially as described in the Promega Technical Bulletin No. 216 usingTfx™-50 to enhance DNA uptake. Typical transfection mixtures contained 5μg of test plasmid (pJ4ΩE6/E7, pJ4ΩE7 or pJ4ΩE6 and pJ4ΩE7), and whererequired 0.1 μg pcDNA3. Where pJ4ΩE6 and pJ4ΩE7 were cotransfected 2.5μg of each was used.

Cells were grown to approximately 80% confluency, the growth mediumremoved and plasmid DNA mixed with Tfx™-50 in a ratio of 4:1 in MinimalEssential Medium was added and the cells incubated at 37° C.

Following 1-2 hours incubation at 37° C. the transfection mixture wasremoved and fresh growth medium added. After 48 hours incubation at 37°C. transfected cells were removed by trysinization and either assayedfor colony formation in soft agar or incubated for a further 24 hours at37° C. before neomycin selection was applied.

For assay of colony formation in soft agar the trysinized cells wereresuspended at a density of 1-5×10⁵ cells/mL in RPMI 1640 supplementedwith 10% FBS, 2 mM glutamine, 10 mM Hepes and 0.084% NaHCO₃ (RPMI1640+)and containing 0.4% agarose (Seaplaque low gelling temperature, FMCBioproducts, USA) maintained at a temperature of 37° C. Following mixing2.5 mL of this suspension was added to each well of a 6 well tray (Nunc)and allowed to set. The trays were then incubated for a period of 10-14days at 37° C. in an atmosphere of 5% CO₂, prior to counting of coloniesusing an inverted light microscope.

Selection of neomycin resistant colonies was carried out on subconfluentcell monolayers using RPMI 1640+ containing 700 μg/mL neomycin(Geneticin). The monolayers were incubated at 37° C. in an atmosphere of5% CO₂ for 10-14 days prior to counting of neomycin resistant coloniesusing an inverted light microscope. Following counting, the colonieswere dispersed by trysinization and assayed for colony formation in softagar as described above. The results of a neomycin selection experimentfollowing transfection of 3T3 cells with various plasmid constructs arepresented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Construct     No. of neomycin                                                                           Mean no. of cells                                   (+pcDNA3)     resistant colonies                                                                        per colony                                          ______________________________________                                        pJ4ΩE6/E7                                                                             2           10                                                  pJ4ΩE7  4           >65                                                 pJ4ΩE6 + pJ4ΩE7                                                                 11          >66                                                 ______________________________________                                    

These results indicate that the E6/E7 fusion is only weakly transformingin comparison with E7 or E6+E7. Both colony numbers and cell growth forthe E6/E7 fusion were low in comparison with the unfused wild-typesequences. This indicates that the outcome of fusing the E6 and E7sequences is impairment of the ability of these sequences to promotecell transformation.

Persons skilled in this art will appreciate that variations andmodifications may be made to the invention as broadly described herein,other than those specifically described without departing from thespirit and scope of the invention. It is to be understood that thisinvention extends to include all such variations and modifications.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 15                                              - <210> SEQ ID NO 1                                                           <211> LENGTH: 37                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 1                                                           #      37          tatg caccaaaaga gaactgc                                    - <210> SEQ ID NO 2                                                           <211> LENGTH: 28                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 2                                                           #             28   tttc tctacgtg                                              - <210> SEQ ID NO 3                                                           <211> LENGTH: 36                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 3                                                           #       36         agat acacctacat tgcatg                                     - <210> SEQ ID NO 4                                                           <211> LENGTH: 35                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 4                                                           #       35         gttt ctgagaacag atggg                                      - <210> SEQ ID NO 5                                                           <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 5                                                           #                48ggag cccgggatgg ggaatccata tgctgtat                        - <210> SEQ ID NO 6                                                           <211> LENGTH: 32                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 6                                                           #          32      cgta gagtcacact tg                                         - <210> SEQ ID NO 7                                                           <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 7                                                           #           30     ttcc atacaaacta                                            - <210> SEQ ID NO 8                                                           <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 8                                                           #           30     ggat gaaatagatg                                            - <210> SEQ ID NO 9                                                           <211> LENGTH: 801                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(798)                                                    <220> FEATURE:                                                                #Sequence: Gene FusionN: Description of Artificial                            - <400> SEQUENCE: 9                                                           - atg cac caa aag aga act gca atg ttt cag ga - #c cca cag gag cga ccc           48                                                                          Met His Gln Lys Arg Thr Ala Met Phe Gln As - #p Pro Gln Glu Arg Pro           #                 15                                                          - aga aag tta cca cag tta tgc aca gag ctg ca - #a aca act ata cat gat           96                                                                          Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gl - #n Thr Thr Ile His Asp           #             30                                                              - ata ata tta gaa tgt gtg tac tgc aag caa ca - #g tta ctg cga cgt gag          144                                                                          Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gl - #n Leu Leu Arg Arg Glu           #         45                                                                  - gta tat gac ttt gct ttt cgg gat tta tgc at - #a gta tat aga gat ggg          192                                                                          Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Il - #e Val Tyr Arg Asp Gly           #     60                                                                      - aat cca tat gct gta tgt gat aaa tgt tta aa - #g ttt tat tct aaa att          240                                                                          Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Ly - #s Phe Tyr Ser Lys Ile           # 80                                                                          - agt gag tat aga cat tat tgt tat agt ttg ta - #t gga aca aca tta gaa          288                                                                          Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Ty - #r Gly Thr Thr Leu Glu           #                 95                                                          - cag caa tac aac aaa ccg ttg tgt gat ttg tt - #a att agg tgt att aac          336                                                                          Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Le - #u Ile Arg Cys Ile Asn           #           110                                                               - tgt caa aag cca ctg tgt cct gaa gaa aag ca - #a aga cat ctg gac aaa          384                                                                          Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gl - #n Arg His Leu Asp Lys           #       125                                                                   - aag caa aga ttc cat aat ata agg ggt cgg tg - #g acc ggt cga tgt atg          432                                                                          Lys Gln Arg Phe His Asn Ile Arg Gly Arg Tr - #p Thr Gly Arg Cys Met           #   140                                                                       - tct tgt tgc aga tca tca aga aca cgt aga ga - #a acc cag ctg ccc ggg          480                                                                          Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Gl - #u Thr Gln Leu Pro Gly           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - atg cat gga gat aca cct aca ttg cat gaa ta - #t atg tta gat ttg caa          528                                                                          Met His Gly Asp Thr Pro Thr Leu His Glu Ty - #r Met Leu Asp Leu Gln           #               175                                                           - cca gag aca act gat ctc tac tgt tat gag ca - #a tta aat gac agc tca          576                                                                          Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gl - #n Leu Asn Asp Ser Ser           #           190                                                               - gag gag gag gat gaa ata gat ggt cca gct gg - #a caa gca gaa ccg gac          624                                                                          Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gl - #y Gln Ala Glu Pro Asp           #       205                                                                   - aga gcc cat tac aat att gta acc ttt tgt tg - #c aag tgt gac tct acg          672                                                                          Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cy - #s Lys Cys Asp Ser Thr           #   220                                                                       - ctt cgg ttg tgc gta caa agc aca cac gta ga - #c att cgt act ttg gaa          720                                                                          Leu Arg Leu Cys Val Gln Ser Thr His Val As - #p Ile Arg Thr Leu Glu           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - gac ctg tta atg ggc aca cta gga att gtg tg - #c ccc atc tgt tct cag          768                                                                          Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cy - #s Pro Ile Cys Ser Gln           #               255                                                           #        801a tct cat cac cat cac cat cac ta - #a                             Lys Pro Arg Ser His His His His His His                                       #           265                                                               - <210> SEQ ID NO 10                                                          <211> LENGTH: 266                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: Gene FusionN: Description of Artificial                            - <400> SEQUENCE: 10                                                          - Met His Gln Lys Arg Thr Ala Met Phe Gln As - #p Pro Gln Glu Arg Pro         #                 15                                                          - Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gl - #n Thr Thr Ile His Asp         #             30                                                              - Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gl - #n Leu Leu Arg Arg Glu         #         45                                                                  - Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Il - #e Val Tyr Arg Asp Gly         #     60                                                                      - Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Ly - #s Phe Tyr Ser Lys Ile         # 80                                                                          - Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Ty - #r Gly Thr Thr Leu Glu         #                 95                                                          - Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Le - #u Ile Arg Cys Ile Asn         #           110                                                               - Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gl - #n Arg His Leu Asp Lys         #       125                                                                   - Lys Gln Arg Phe His Asn Ile Arg Gly Arg Tr - #p Thr Gly Arg Cys Met         #   140                                                                       - Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Gl - #u Thr Gln Leu Pro Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met His Gly Asp Thr Pro Thr Leu His Glu Ty - #r Met Leu Asp Leu Gln         #               175                                                           - Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gl - #n Leu Asn Asp Ser Ser         #           190                                                               - Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gl - #y Gln Ala Glu Pro Asp         #       205                                                                   - Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cy - #s Lys Cys Asp Ser Thr         #   220                                                                       - Leu Arg Leu Cys Val Gln Ser Thr His Val As - #p Ile Arg Thr Leu Glu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cy - #s Pro Ile Cys Ser Gln         #               255                                                           - Lys Pro Arg Ser His His His His His His                                     #           265                                                               - <210> SEQ ID NO 11                                                          <211> LENGTH: 519                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(516)                                                    <220> FEATURE:                                                                #Sequence: Gene FusionN: Description of Artificial                            - <400> SEQUENCE: 11                                                          - atg ggg aat cca tat gct gta tgt gat aaa tg - #t tta aag ttt tat tct           48                                                                          Met Gly Asn Pro Tyr Ala Val Cys Asp Lys Cy - #s Leu Lys Phe Tyr Ser           #                 15                                                          - aaa att agt gag tat aga cat tat tgt tat ag - #t ttg tat gga aca aca           96                                                                          Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Se - #r Leu Tyr Gly Thr Thr           #             30                                                              - tta gaa cag caa tac aac aaa ccg ttg tgt ga - #t ttg tta att agg tgt          144                                                                          Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys As - #p Leu Leu Ile Arg Cys           #         45                                                                  - att aac tgt caa aag cca ctg tgt cct gaa ga - #a aag caa aga cat ctg          192                                                                          Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Gl - #u Lys Gln Arg His Leu           #     60                                                                      - gac aaa aag caa aga ttc cat aat ata agg gg - #t cgg tgg acc ggt cga          240                                                                          Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gl - #y Arg Trp Thr Gly Arg           # 80                                                                          - tgt atg tct tgt tgc aga tca tca aga aca cg - #t aga gaa acc cag ctg          288                                                                          Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Ar - #g Arg Glu Thr Gln Leu           #                 95                                                          - ccc ggg atg cat gga gat aca cct aca ttg ca - #t gaa tat atg tta gat          336                                                                          Pro Gly Met His Gly Asp Thr Pro Thr Leu Hi - #s Glu Tyr Met Leu Asp           #           110                                                               - ttg caa cca gag aca act gat ctc tac tgt ta - #t gag caa tta aat gac          384                                                                          Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Ty - #r Glu Gln Leu Asn Asp           #       125                                                                   - agc tca gag gag gag gat gaa ata gat ggt cc - #a gct gga caa gca gaa          432                                                                          Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pr - #o Ala Gly Gln Ala Glu           #   140                                                                       - ccg gac aga gcc cat tac aat att gta acc tt - #t tgt tgc aag tgt gac          480                                                                          Pro Asp Arg Ala His Tyr Asn Ile Val Thr Ph - #e Cys Cys Lys Cys Asp           145                 1 - #50                 1 - #55                 1 -       #60                                                                           #    519g ctt cgg aga tct cat cac cat cac ca - #t cac taa                     Ser Thr Leu Arg Arg Ser His His His His Hi - #s His                           #               170                                                           - <210> SEQ ID NO 12                                                          <211> LENGTH: 172                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: Gene FusionN: Description of Artificial                            - <400> SEQUENCE: 12                                                          - Met Gly Asn Pro Tyr Ala Val Cys Asp Lys Cy - #s Leu Lys Phe Tyr Ser         #                 15                                                          - Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Se - #r Leu Tyr Gly Thr Thr         #             30                                                              - Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys As - #p Leu Leu Ile Arg Cys         #         45                                                                  - Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Gl - #u Lys Gln Arg His Leu         #     60                                                                      - Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gl - #y Arg Trp Thr Gly Arg         # 80                                                                          - Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Ar - #g Arg Glu Thr Gln Leu         #                 95                                                          - Pro Gly Met His Gly Asp Thr Pro Thr Leu Hi - #s Glu Tyr Met Leu Asp         #           110                                                               - Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Ty - #r Glu Gln Leu Asn Asp         #       125                                                                   - Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pr - #o Ala Gly Gln Ala Glu         #   140                                                                       - Pro Asp Arg Ala His Tyr Asn Ile Val Thr Ph - #e Cys Cys Lys Cys Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Thr Leu Arg Arg Ser His His His His Hi - #s His                         #               170                                                           - <210> SEQ ID NO 13                                                          <211> LENGTH: 519                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(519)                                                    <220> FEATURE:                                                                #Sequence: Gene FusionN: Description of Artificial                            - <400> SEQUENCE: 13                                                          - atg gag gag gat gaa ata gat ggt cca gct gg - #a caa gca gaa ccg gac           48                                                                          Met Glu Glu Asp Glu Ile Asp Gly Pro Ala Gl - #y Gln Ala Glu Pro Asp           #                 15                                                          - aga gcc cat tac aat att gta acc ttt tgt tg - #c aag tgt gac tct acg           96                                                                          Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cy - #s Lys Cys Asp Ser Thr           #             30                                                              - ctt cgg ttg tgc gta caa agc aca cac gta ga - #c att cgt act ttg gaa          144                                                                          Leu Arg Leu Cys Val Gln Ser Thr His Val As - #p Ile Arg Thr Leu Glu           #         45                                                                  - gac ctg tta atg ggc aca cta gga att gtg tg - #c ccc atc tgt tct cag          192                                                                          Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cy - #s Pro Ile Cys Ser Gln           #     60                                                                      - aaa cca gga tct cat atg cac caa aag aga ac - #t gca atg ttt cag gac          240                                                                          Lys Pro Gly Ser His Met His Gln Lys Arg Th - #r Ala Met Phe Gln Asp           # 80                                                                          - cca cag gag cga ccc aga aag tta cca cag tt - #a tgc aca gag ctg caa          288                                                                          Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Le - #u Cys Thr Glu Leu Gln           #                 95                                                          - aca act ata cat gat ata ata tta gaa tgt gt - #g tac tgc aag caa cag          336                                                                          Thr Thr Ile His Asp Ile Ile Leu Glu Cys Va - #l Tyr Cys Lys Gln Gln           #           110                                                               - tta ctg cga cgt gag gta tat gac ttt gct tt - #t cgg gat tta tgc ata          384                                                                          Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Ph - #e Arg Asp Leu Cys Ile           #       125                                                                   - gta tat aga gat ggg aat cca tat gct gta tg - #t gat aaa tgt tta aag          432                                                                          Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cy - #s Asp Lys Cys Leu Lys           #   140                                                                       - ttt tat tct aaa att agt gag tat aga cat ta - #t tgt tat agt ttg tat          480                                                                          Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Ty - #r Cys Tyr Ser Leu Tyr           145                 1 - #50                 1 - #55                 1 -       #60                                                                           #    519a aca tta aga tct cat cac cat cac ca - #t cac taa                     Gly Thr Thr Leu Arg Ser His His His His Hi - #s His                           #               170                                                           - <210> SEQ ID NO 14                                                          <211> LENGTH: 172                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: Gene FusionN: Description of Artificial                            - <400> SEQUENCE: 14                                                          - Met Glu Glu Asp Glu Ile Asp Gly Pro Ala Gl - #y Gln Ala Glu Pro Asp         #                 15                                                          - Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cy - #s Lys Cys Asp Ser Thr         #             30                                                              - Leu Arg Leu Cys Val Gln Ser Thr His Val As - #p Ile Arg Thr Leu Glu         #         45                                                                  - Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cy - #s Pro Ile Cys Ser Gln         #     60                                                                      - Lys Pro Gly Ser His Met His Gln Lys Arg Th - #r Ala Met Phe Gln Asp         # 80                                                                          - Pro Gln Glu Arg Pro Arg Lys Leu Pro Gln Le - #u Cys Thr Glu Leu Gln         #                 95                                                          - Thr Thr Ile His Asp Ile Ile Leu Glu Cys Va - #l Tyr Cys Lys Gln Gln         #           110                                                               - Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Ph - #e Arg Asp Leu Cys Ile         #       125                                                                   - Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cy - #s Asp Lys Cys Leu Lys         #   140                                                                       - Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Ty - #r Cys Tyr Ser Leu Tyr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Thr Thr Leu Arg Ser His His His His Hi - #s His                         #               170                                                           - <210> SEQ ID NO 15                                                          <211> LENGTH: 12                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence: PrimerMATION: Description of Artificial                            - <400> SEQUENCE: 15                                                          #       12                                                                    __________________________________________________________________________

We claim:
 1. An isolated variant human papillomavirus (HPV) protein ableto elicit an immune response against HPV in a host animal but not beingcell-transforming in said host animal, wherein said variant proteincomprises a fusion protein comprising a HPV E6 protein selected from thegroup consisting of full length E6 protein and non-full length deletionmutants thereof, and a HPV E7 protein selected from the group consistingof full length E7 protein and non-full length deletion mutants thereof,and optionally a linker linking said E6 and E7 proteins.
 2. An isolatedvariant HPV protein according to claim 1, wherein said protein furthercomprises a foreign protein or peptide fused or otherwise coupled to oneor both of said E6 and E7 proteins.
 3. An isolated variant HPV proteinaccording to claim 2, wherein the foreign protein or peptide is selectedfrom the group consisting of (i) proteins or peptides to assist inpurification of the fusion protein or (ii) proteins or peptides toenhance the immunogenicity of the fusion protein.
 4. An isolated variantHPV protein acording to claim 1, which comprises a fusion proteincomprising a full length E6 protein fused to a full length E7 protein.5. An isolated variant HPV protein according to claim 4, which comprisesthe full length E6 protein as an N-terminal sequence of the fusionprotein and the full length E7 protein as a C-terminal sequence of thefusion protein.
 6. An isolated variant HPV protein according to claim 4,which comprises the full length E7 protein as an N-terminal sequence ofthe fusion protein and the full length E6 protein as a C-terminalsequence of the fusion protein.
 7. An isolated variant HPV proteinaccording to claim 1, which is a fusion protein comprising a non-fulllength deletion mutant of the E6 protein as an N-terminal sequence ofthe fusion protein and a non-full length deletion mutant of the E7protein as a C-terminal sequence of the fusion protein.
 8. An isolatedvariant HPV protein according to claim 1, which is a fusion proteincomprising a non-full length deletion mutant of the E7 protein as anN-terminal sequence of the fusion protein and a non-full length deletionmutant of the E6 protein as a C-terminal sequence of the fusion protein.9. An isolated variant HPV protein according to claim 1, wherein thenon-full length deletion mutants of the E6 or E7 proteins comprise atleast 50% of the full length sequences of the proteins.
 10. An isolatedvariant HPV protein according to claim 9, wherein the non-full lengthdeletion mutants of the E6 or E7 protein comprise at least 60% of thefull-length sequences of the proteins.
 11. An isolated variant HPVprotein according to claim 10, wherein the non-full length deletionmutants of the E6 or E7 proteins comprise at least the N-terminal 60% ofthe full length sequence of the proteins.
 12. An isolated variant HPVprotein according to claim 10, wherein the non-full length deletionmutants of the E6 or E7 proteins comprise at least the C-terminal 60% ofthe full length sequence of the proteins.
 13. An isolated variant HPVprotein according to claim 1, wherein said linker consists of from 1 to50 amino acid residues.
 14. An isolated variant HPV protein according toclaim 13, wherein said linker consists of from 1 to 20 amino acidresidues.
 15. An isolated variant HPV protein according to claim 4,wherein said linker consists of from 1 to 5 amino acid residues.
 16. Anisolated variant HPV protein according to claim 1, wherein said E6 or E7protein is selected from the group consisting of HPV-16, HPV-18, HPV-6and HPV-11 genotypes.
 17. An isolated variant HPV protein according toclaim 16, wherein said E6 or E7 protein is selected from the groupconsisting of HPV-16 and HPV-18 genotypes.
 18. A composition for use ineliciting an immune response against HPV in a host animal, saidcomposition comprising an isolated variant HPV protein acording to claim1, together with a pharmaceutically acceptable carrier and/or diluent.19. A composition according to claim 18, further comprising an adjuvant.20. A method for eliciting an immune response against HPV in a hostanimal, which method comprises administering to the host animal aneffective amount of an isolated variant HPV protein according toclaim
 1. 21. A method according to claim 20, wherein said variant HPVprotein is administered in a composition together with apharmaceutically acceptable carrier and/or diluent.
 22. A methodaccording to claim 21, wherein said composition further comprises anadjuvant.
 23. A method according to claim 20, wherein said host animalis a human.